Study On Anti-HBV Active Fractions Of The Ascidian Styela Plicata (Lesueur) In Vitro

Hepatitis B virus is a big danger for human's health. There are 120 millionHBV carriers and 30 million type B hepatitis (HB) patients in China. People infectedHBV have a high dead risk for hepatic failure, hepatic cirrhosis or primary liver cellcancer. It is necessary and imperative to find some novel and high effective drugsfrom Chinese traditional herbs and marine drugs to cure the HB and control the highpopular of HBV.Styela plicata (lesueur) is an ascidian which belongs to class Ascidiacea,subphylum urochordata. More than 1500 kinds of ascidians have been known in thenature and they mainly distribute over the tropical zone and subtropical zone. Thereare abundant ascidian resources in the South China Sea. Studying on bioactivecomponents from ascidian is a popular domain in marine drugs research during theseyears. Many chemical compositions such as alkaloids, peptide, benzopyrrole, heavymetal chelating reagent, polysulfide, macrolide, terpenoid, and so on, have beenfound in ascidian. And most of them have strong biological activity, such asanti-tumor, anti-virus, antibiosis, and sarcoplasmic reticulum to dispel calcium, etc.At present ascidian research is concentrated on anti-tumor and anti-viruscompositions separation, synthesis and reconstruction. But there are few reportsabout anti-HBV compositions from ascidian. According to the reports aboutalkaloids and saponins extracted from ascidian, we carried on series trials to detect the anti-HBV effect of alkaloids and saponins from Styela plicata (lesueur) usingHepG2 2.2.15 cell transfected HBV as screening model in vitro. A HPLC fingerprinting of anti-HBV effective fractions from Styela plicata has also been established,and it could be used to identify the sources of Styela plicata and control the qualityof ascidian's preparations.This report has three parts as below:1 Separation of effective fractions from Styela plicata (lesueur)We acquired the alkaloids and n-butanol fractions respectively using thegeneral isolation design from the Fresh Styela plicata. The major isolation processare as below: Fresh Styela plicata (with entrails stripped) was soaked in 95% alcohol(using H₂SO₄ to adjust pH=3) for 24h and was recycled-extracted for 2h. Then theleachate was filtrated and the extracts was obtained by evaporating the solvent. Afterthat, the extracts was resolved in water (containing 1% H₂SO₄) and the watersolution was extracted by equal volume of chloroform. The remain water wasadjusted to pH=9.5 by 10% ammonia water, and the alkalia water was extracted byequal volume of chloroform again. At last the alkaloids were obtained byevaporating the chloroform part. The n-butanol fraction(S) extracted from Styelaplicata were systematic separated by gel silica chromatography using chloroformand methanol as eluant. The Fr-S1, Fr-S2, Fr-S3 and Fr-S4 were collected, and thesubstance HQS3 acquired from the Fr-S3 showed a strongly anti-HBV effect.2 Anti-HBV effect assay of the effective fractions extracted from Styela plicata(lesueur)The anti-HBV effect of the alkaloids and n-butanol fractions were evaluated bythe HepG2 2.2.15 cell model in vitro. According to the pharmacodynamic and MTTresults, the alkaloids had a strong inhibition on the HepG2 2.2.15 cell to excrete theHBsAg and HBeAg, the IC₅₀ was 1.110mg/ml, 1.715mg/ml respectively, and thealkaloids have no cytotoxicity through the MTT dates. Through the trials, then-butanol fraction(S), Fr-S2 and Fr-S3 had anti-HBV effects, the Fr-S3 revealed afierce inhibition of HepG2 2.2.15 cell to secrete the HBsAg and HBeAg. So, we takea deep separation of Fr-S3 and the HQS3 was obtained by the gel silicachromatography. The HQS3 had a strong anti-HBV effect at a low concentration(0.125mg/ml). IC₅₀ (HBsAg) of HQS3 was 0.159mg/ml and IC₅₀(HBeAg) was 0.468mg/ml. HQS3 showed some toxicity in high concentration, but the rate ofdamage was low than 50% and their TC₅₀ were more than 0.5mg/ml. Its TI (HBsAg)were more than 3.41and TI (HBeAg) was more than 1.07. On the basis of its TI, theHQS3 can be presumed to have inhibitory effect on HBsAg and HBeAg expressionin HepG2 2.2.15 cell and have little cytotoxicity. This conclusion would give aguideline for the research on anti-HBV drugs.3 Study on finger printing of anti-HBV effective fraction of Styela plicataHPLC technology was used to establish finger printing of anti-HBV effectivefraction of Styela plicata. This method was on the base of effective purepolypeptide-Sub (Ser-Ser-Leu-Ser-Lys-Ala-Ala) separated from Styela plicata. Andanti-HBV effective fraction of Styela plicata was the quality control target. Lineargradient elution was used in this experiment and the chromatographic conditioncould be controlled easily. Every peak was separable and the result had goodprecision and repeatability.Thirteen common chromatographic peaks of this anti-HBV effective fractioncould be detected on this chromatographic condition. In the figure, chromatographicpeak S which was regarded as reference peak was the peak of Sub. Contrasting tothis reference peak, relative retention time and peak area ratio could be calculated.Relative retention time of thirteen peaks was rather stable in HPLC figures. Theaverage relative retention time of ten samples were 0.95(1),1.00(S),1.21(3),1.28(4),1.54(5),1.61(6),1.71(7),1.78(8),1.85(9),1.98(10),2.05(11),2.10(12),2.28(13), respectively. And relative standard deviation was less than 3%. Theaverage relative retention time could be considered as a quality identification index.The average relative common peaks area ratio of ten samples were 0.27(1),1.00(S),0.45(3),0.98(4),5.04(5),1.78(6),0.85(7),0.44(8),0.75(9),0.76(10),0.18(11),0.17(12),0.95(13), respectively, and the areas of thirteen common peaks take upmore than 90% of total areas. All those results are compliant with guideline for thefingerprint of Chinese traditional drugs. As key feature peaks in finger printing, theycould be used to identify the sources of Styela plicata and control the quality ofascidian's preparations.